Method of screening anti-mycobacterial molecules

ABSTRACT

This invention relates to a novel mycobacterial protein named DES, which appears to share significant amino acid sequence homology with soluble stearoyl-ACP desaturases. The results of allelic exchange experiments, indicate that the des gene may be essential to the survival of mycobacteria. These results coupled with the surface localization, the unique structure of DES, and the fact this antigen is expressed in vivo, and DES protein induces a humoral response in human patients, indicate that the DES protein provides a new target for the design of anti-mycobacterial drugs. This invention provides methods of screening molecules that can inhibit the DES enzyme activity of purified DES protein, in order to identify antibiotic molecules that are capable of inhibiting the growth or survival of mycobacteria. These methods may be practiced by using recombinant DES protein obtained from a recombinant mycobacterium host cell that was transformed with a vector containing the des gene, whose expression is controlled by regulatory or promoter sequences that function in mycobacteria. Another aspect of this invention relates to the molecules that have been identified according to the screening methods as having antibiotic activity against mycobacteria.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application hereby claims the benefit under 35 U.S.C.§119(e) of U.S. provisional applications S. No. 60/113,375, filed Nov.4, 1998; S. No. 60/111,813, filed Dec. 11, 1998; and U.S. applicationSer. No. 09/181,934, filed Oct. 28, 1998, which was converted to aprovisional application under 37 C.F.R. §1.53(c)(2)on Jan. 14, 1999. Theentire disclosure of each of these applications is relied upon andincorporated by reference herein.

BACKGROUND OF THE INVENTION

[0002] Tuberculosis and leprosy, caused by the bacilli from theMycobacterium tuberculosis complex and M. leprae, respectively, are thetwo major mycobacterial diseases. Other mycobacteriosis caused by atypical mycobacteria such as M. avium, M. xenopi, and M. Kansasii alsorepresent major health problems worldwide.

[0003]M. avium is a predominant strain isolated from T.B. patients withAIDS (Horburgh et al., 1991) and M. xenopi along with M. kansasii and M.avium, is the main agent of pulmonary infections due to opportunistmycobacteria in HIV seronegative patients. (M. Picardeau et al., 1995).

[0004] In addition, these a typical mycobacteriosis are often difficultto cure because of the lack of efficient drugs specifically directedagainst a typical mycobacteria. Pathogenic mycobacteria have the abilityto survive within host phagocytic cells. The pathology of thetuberculosis infection derives from the interactions between the hostand the bacteria, resulting from the damage the host immune responsecauses on tissues (Andersen & Brennan, 1994). In addition, theprotection of the host against mycobacteria infection also depends oninteractions between the host and mycobacteria.

[0005] Identification of the bacterial antigens involved in theseinteractions with the immune system is essential for the understandingof the pathogenic mechanisms of mycobacteria and the host immunologicalresponse in relation to the evolution of the disease. It is also ofgreat importance for the improvement of the strategies for mycobacterialdisease control through vaccination and immunodiagnosis.

[0006] Through the years, various strategies have been followed foridentifying mycobacterial antigens. Biochemical tools for fractionatingand analyzing bacterial proteins permitted the isolation of antigenicproteins selected on their capacity to elicit B- or T-cell responses(Romain et al., 1993; Sorensen et al., 1995). The recent development ofmolecular genetic methods for mycobacteria (Jacobs et al., 1991; Snapperet al., 1990; Hatful, 1993; Young et al., 1985) allowed the constructionof DNA expression libraries of both M. tuberculosis and M. leprae in theλgt11 vector and their expression in E. coli. The screening of theserecombinant libraries using murine polyclonal or monoclonal antibodiesand patient sera led to the identification of numerous antigens(Braibant et al., 1994; Hermans et al., 1995; Thole & van der Zee,1990). However, most of them turned out to belong to the group of highlyconserved heat shock proteins (Thole & van der Zee, 1990; Young et al.,1990).

[0007] The observation in animal models that specific protection againsttuberculosis was conferred only by administration of live BCG vaccine,suggested that mycobacterial secreted proteins might play a major rolein inducing protective immunity. These proteins were shown to inducecell-mediated immune responses and protective immunity in a guinea pigor a mouse model of tuberculosis (Pal & Horwitz, 1992; Andersen, 1994;Haslov et al., 1995). Recently, a genetic methodology for theidentification of exported proteins based on PhoA gene fusions wasadapted to mycobacteria by (Lim et al., 1995). It permitted theisolation of M. tuberculosis DNA fragments encoding exported proteins,including the already known 19 kDa lipoprotein (Lee et al., 1992) andthe ERP protein similar to the M. leprae 28 kDa antigen (Berthet et al.,1995).

SUMMARY OF THE INVENTION

[0008] We have characterized a new M. tuberculosis exported proteinnamed DES, identified by using the PhoA gene fusion methodology. The desgene, which seems conserved among mycobacterial species, encodes anantigenic protein highly recognized by human sera from both tuberculosisand leprosy patients but not by sera from tuberculous cattle. Theresults of allelic exchange experiments described in this application,indicate that the des gene is essential to the survival of mycobacteria.

[0009] The amino acid sequence of the DES protein contains two sets ofmotifs that are characteristic of the active sites of enzymes from theclass II diiron-oxo protein family. Among this family, the DES proteinpresents significant homologies to soluble stearoyl-acyl carrier protein(ACP) desaturases. Three dimensional modeling demonstrates that the DESprotein and the plant stearoyl-ACP desaturase share a conserved activesite.

[0010] This invention also provides methods of identifying moleculescapable of inhibiting the growth and/or survival of Mycobacteriaspecies. In particular, the methods of this invention include screeningmolecules that can inhibit the activity of the DES protein. Thesemethods comprise the steps of:

[0011] a) contacting the molecule with a strain of mycobacteria speciescontaining an active DES protein or a DES like protein or a vectorcarrying an active DES protein gene or a vector containing apolynucleotide sequence encoding the active site of the DES protein;

[0012] b) measuring the inhibition of the growth of said mycobacteriastrain; and

[0013] c) identifying the molecule that is reacting with the DES proteinor with the active site of said protein carrying conserved residues.

[0014] To practice the methods of this invention, the purified DESprotein may be a recombinant desaturase protein. The recombinant DESprotein can be obtained from a recombinant mycobacterium host cell thatwas transformed with an expression vector containing a polynucleotideencoding the DES protein whose expression is controlled by regulatorysequences that function in mycobacteria.

[0015] In one method of the invention, the recombinant expression vectoris a plasmid derived from the pJAM2 plasmid (e.g. pJAM21). The inventionalso encompasses the pJAM2 and pJAM21 plasmids, as well as recombinanthost cells transformed with the pJAM2 and pJAM21 plasmids. A recombinanthost cell transformed with pJAM21 has been deposited at CollectionNationale de Cultures de Micro-organisms (CNCM) in Paris, France, onJun. 23, 1998, under accession number I-2042.

[0016] Another aspect of this invention relates to molecules that havebeen screened according to the methods of this invention and identifiedas having antibiotic activity against mycobacteria.

[0017] It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

[0018] The accompanying drawings, which are incorporated in andconstitute a part of this specification, illustrate several embodimentsof the invention and together with description, serve to explain theprinciples of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 is a restriction map of the 4.5 kb EcoRV fragment encodingthe M. tuberculosis des gene.

[0020]FIG. 2A is a vector map for the pJAM2 plasmid.

[0021]FIG. 2B is the nucleotide sequence of the multi-cloning site andsurrounding regions of pJAM2. The Shine-Delgarno sequence (S.D.) isshown in bold type.

[0022]FIG. 3 shows a comparative sequence analysis of class IIdiiron-oxo proteins and the M. tuberculosis DES protein. Shaded residuesindicate cluster ligands and probable iron ligands in the M.tuberculosis DES protein. Bold unshaded framed letters are probableresidues involved in the network of hydrogen bonds to the cluster. Otherbold letters indicate conserved residues that are believed toparticipate in the O₂-binding site. Gaps introduced into the sequence ofDES are indicated by dots.

[0023] Accession numbers are as follows: V015555, Epstein-Barr virusribonucleotide reductase; M58499, Methylococcus capsulatus methanemono-oxygenase hydroxylase; M60276, Pseudomonas sp. strain CF 600 phenolhydroxylase dmpN polypeptide; M59857, Ricinus communis stearoyl-ACPdesaturase; and D38753, O. sativa stearoyl-ACP desaturase.

[0024]FIG. 4 is a Southern blot analysis of the distribution of the desgene in other mycobacterial species. DNA from various mycobacterialstrains were PstI-digested, electrophoresed, transferred onto a nylonmembrane by Southern blotting, and hybridized using probe B, which isshown in FIG. 1.

[0025]FIG. 5 shows an SDS-PAGE gel of soluble and insoluble extractsfrom E. coli expressing the DES protein on plasmid pETdes (I-1718).

[0026]FIG. 6 shows the results of ELISAs of the sensitivity of theantibody response to the DES antigen of human tuberculous andnon-tuberculous patients.

[0027]FIG. 7 shows the nucleotide and derived amino acid sequences ofthe Mycobacterium tuberculosis des gene. The underlined sequencescorrespond to the −35 and −10 boxes of the promoter and a Shine Delgarnosequence that corresponds to the putative ribosomal attachment site,respectively. The adenosine labeled “+1” corresponds to thetranscription initiation site.

[0028]FIG. 8 is a table of the bacterial strains and plasmids used inthis application.

[0029]FIG. 9 is a Western blot showing the recognition of the purifiedDES protein by antibodies from M. bovis and M. tuberculosis-infectedhumans and cattle.

[0030]FIG. 10 shows the inducible expression of the gene encoding the M.leprae 35 kDa protein in M. smegmatis in the presence or absence of theacetamidase inducer acetamide. Section (A) is an SDS-PAGE gel ofbacterial sonicates and purified protein. Section (B) is a Western blotof a corresponding gel analyzing reactivity with the anti-M. leprae mAbCS38. Lane 1 corresponds to M. smegmatis harboring pJAM4 grown in theabsence of acetamide; lane 2 corresponds to M. smegmatis harboring pJAM4grown in the presence of acetamide; lane 3 corresponds to purified M.leprae 35 kDa protein.

[0031]FIG. 11 is a table representing the quantification of the M.leprae protein produced in recombinant M. smegmatis in the presence orabsence of the acetamidase inducer acetamide. Results are expressed asthe mean value±SEM of three experiments. Suc: is an abbreviation forsuccinate; Suc/Act: is an abbreviation succinate plus acetamide.

[0032]FIG. 12 is a graph representing the recognition of the recombinantM. leprae 35 kDa protein by lepromatous leprosy sera. In the legend, M.smg 35 kDa: represents M. smegmatis-derived 35 kDa protein; M. smg 35kDa-HIS: represents M. smegmatis-derived, histidine-tagged 35 kDaprotein; and E. coli 35 kDa: represents E. coli-derived 35 kDa protein.

[0033]FIG. 13 is a Western blot showing induction of the gene encodingthe M. tuberculosis DES antigen in M. smegmatis using the pJAM2expression system. Ten μg of cell sonicate from 1 bacteria grown in theabsence (−) or presence (+) of acetamide were added to each lane and thetransferred gel was immunoblotted with anti-DES murine polyclonalantibody. WT represents wild-type M. smegmatis mc²155; and MYC1553represents M. smegmatis harboring pJAM21. Sonicates from twotranformants are shown. The location of the DES antigen is indicated.

DETAILED DESCRIPTION

[0034] Using the Pho A gene fusion methodology, we identified a new 37kDa Mycobacterium tuberculosis protein, designated DES. This 37 kDaexported protein contains conserved amino acid residues which arecharacteristic of class II diiron-oxoproteins. Proteins from that familyare all enzymes that require iron for activity. They includeribonucleotide reductases, hydrocarbon hydroxylases and stearoyl-ACPdesaturases. The M. tuberculosis DES protein only presents significanthomologies to plant stearoyl-ACP desaturases (44% identity at thenucleotide level, and 30% identity at the amino-acid level), which areexported enzymes as they are translocated across the chloroplasticmembranes (Keegstra & Olsten, 1989).

[0035] Three-dimensional modeling of the DES protein based on homologywith the Ricinus communis Δ9 stearoyl-ACP indicates that the DES proteinshares significant structural features with the plant stearoyl-ACPdesaturases. Most importantly, the active site of the DES protein andthe plant Δ9 stearoyl-ACP desaturase are conserved, suggesting that DESis evolutionarily related to the plant desaturases.

[0036] The plant stearoyl-ACP desaturase can be used for the screeningand the selection of new compounds inhibiting the activity of the enzymeand consequently then tested for the modulation of the properties of DESprotein in vivo in a mycobacterial strain, such as M. tuberculosis or invitro on a purified DES protein. This result suggests that the DESprotein could be involved in the mycobacterial fatty acid biosynthesis.

[0037] Furthermore, the localization of the protein outside thecytoplasm would be consistent with its role in the lipid metabolism,since lipids represent 60% of the cell wall constituents and that partof the biosynthesis of the voluminous mycolic acids containing 60 to 90carbon atoms occurs outside the cytoplasm. Among all the different stepsof the lipid metabolism, desaturation reactions are of special interest,first because they very often take place at early steps of lipidbiosynthesis and secondly because, through the control they have on theunsaturation rate of membranes, they contribute to the adaptation ofmycobacteria to their environment (Wheeler & Ratledge, 1994). An enzymesystem involving a stearoyl-Coenzyme A desaturase (analog of the plantstearoyl-ACP-desaturases), catalyzing oxydative desaturation of the CoAderivatives of stearic and palmitic acid to the corresponding Δ9monounsaturated fatty acids has been biochemically characterized inMycobacterium phlei (Fulco & Bloch, 1962; Fulco & Bloch, 1964;Kashiwabara et al., 1975; Kashiwabara & Sato, 1973). This system wasshown to be firmly bound to a membranous structure (Fulco & Bloch,1964). Thus, M. tuberculosis stearoyl-Coenzyme A desaturase (Δ9desaturase) is expected to be an exported protein.

[0038] Sonicated extracts of E. coli expressing the DES protein wereassayed for Δ9 desaturating activity according to the method describedby (Legrand and Bensadoun, 1991), using (stearoyl-CoA) ¹⁴C as asubstrate. However, no Δ9 desaturating activity could be detected. Thisresult is probably linked to the fact that desaturation systems aremulti-enzyme complexes involving electron transport chains and numerouscofactors, often difficult to render functional in vitro. Since E.coliand mycobacteria are very different from a lipid metabolism point ofview, in E. coli, the M. tuberculosis recombinant Δ9 desaturase mightnot dispose of all the cofactors and associated enzymes required foractivity or might not interact properly with them. Moreover, not allcofactors involved in the Δ9 desaturation process of mycobacteria areknown, and they might be missing in the incubation medium.

[0039] However, if the DES protein encodes a Δ9 desaturase, aninteresting point concerns its primary sequence. Indeed, all animal,fungal, and the only two bacterial Δ9 desaturases sequenced to date(Sakamoto et al., 1994) are integral membrane proteins which have beenclassified into a third class of diiron-oxo proteins on the basis oftheir primary sequences involving conserved histidine residues (Shanklinet al., 1994). The plant soluble Δ9 desaturases are the only desaturasesto possess the type of primary sequence of class II diiron-oxo proteins(Shanklin & Somerville, 1991). No bacteria have yet been found whichhave a plant type Δ9 desaturase.

[0040] As shown by immunoblotting and ELISA experiments, the DES proteinis a highly immunogenic antigen which elicits a B-cell response in 100%of the tuberculosis M. bovis or M. tuberculosis-infected human patientstested, independently of the form of the disease (extrapulmonary orpulmonary). It also elicits an antibody response in lepromatous leprosypatients. Interestingly, although more sera would need to be tested,tuberculous cattle do not seem to recognize the DES antigen.Furthermore, the ELISA experiments showed that it is possible todistinguish tuberculosis patients from patients suffering from otherpathologies on the basis of the sensitivity of their antibody responseto the DES antigen. The DES antigen is therefore a good candidate to beused for serodiagnosis of tuberculosis in human patients.Non-tuberculous patients may recognize the DES protein at a low levelbecause they are all BCG-vaccinated individuals (BCG expressing theprotein), or because of cross-reactivity of their antibody response withother bacterial antigens. It would now be interesting to know whetherthe DES antigen possesses in addition to its B-cell epitopes, T-cellepitopes, which are the only protective epitopes in the hostimmunological response against pathogenic mycobacteria. If the DESprotein is also a good stimulator of the T-cell response in a majorityof tuberculosis patients, it could be used either individually or aspart of a cocktail of antigens in the design of a subunit vaccineagainst tuberculosis.

[0041] To gain insights into the precise function of this a typicalbacterial enzyme, we attempted to interrupt the des gene in the vaccinestrain M. bovis BCG by allelic exchange. In a first experiment, noallelic exchange mutants were obtained, suggesting that the des gene isessential to the viability of mycobacteria. To investigate thishypothesis, the first experiment was repeated using a M. bovis BCGstrain transformed with a second wild-type copy of the des gene. Usingthis transformed M. bovis BCG strain, we obtained allelic exchangemutants, in which a wild-type copy of the des gene was replaced by aninactivated copy of the des gene. Thus, allelic exchange was onlypossible if a second copy of the wild-type des gene had been insertedinto the M. bovis BCG chromosome. This result strongly suggests that desis an essential gene in mycobacteria from the M. tuberculosis complex.

[0042] Coupled with the localization of DES at the surface of thetubercle bacilli, and its structural originality (this enzyme'sstructure differs from all the mammalian and bacterial desaturasestructures identified to date), the results of these experiments suggestthat the DES protein could be a target for designing newanti-mycobacterial drugs.

[0043] Fundamental to the analysis of the biological function andimmunological relevance of mycobacterial proteins is their production ina recombinant form that resembles that of their native counterpart.Recent studies analyzing both structure (Garbe et al., 1993; Triccas etal., 1996) and immunogenicity (Garbe et al., 1993; Roche et al., 1996;Triccas et al., 1996) of recombinant proteins obtained from fast growingmycobacterial hosts, such as Mycobacterium smegmatis, have demonstratedsuperiority over the same protein purified from E. coli expressionsystems. Although such approaches for the production of recombinantmycobacterial proteins appear advantageous, two major obstacles lie inthe way of further improvement to these systems. The first is theinability to regulate high-level expression of foreign genes in M.smegmatis, analogous to systems such as induction of the lac promoter inE. coli (de Boer et al., 1983). Secondly, no simple, efficient andwidely adaptable method for the purification of proteins fromrecombinant mycobacteria has been described.

[0044] In this application, we attempt to resolve these two problems.First, we describe the construction of a vector, pJAM2, that utilizesthe promoter of the inducible acetamidase enzyme of M. smegmatis todrive high-level expression of foreign genes in M. smegmatis. The 47 kDaacetamidase enzyme of M. smegmatis NCTC 8159 permits the growth of theorganism on simple amides as the sole carbon source and is highlyinducible in the presence of acetamide (Mahenthiralingam et al., 1993).This property has been previously used to assess luciferase as areporter of gene expression in mycobacteria (Gordon et al., 1994) and todevelop a mycobacterial-conditional antisense mutagenesis system (Parishet al., 1997b). In this study, we constructed a vector that allows forregulated high-level expression of foreign genes in mycobacteria byvirtue of the M. smegmatis acetamidase promoter.

[0045] Recombinant M. leprae 35 kDa antigen produced in this systemrepresented approximately 8.6% of the total M. smegmatis solubleprotein, with the amount of protein produced greater than that when thesame gene is placed under the control of the strong mutated β-lactamasepromoter of M. fortuitum (FIG. 3).

[0046] Secondly, we demonstrate the simple and efficient purification ofthe encoded antigens by use of a poly-histidine tag and one step Ni⁺⁺affinity chromatography. The addition of the histidine tag did notappear to affect the conformation or immunogenicity of the recombinantprotein, suggesting the system described may be extremely useful for thepurification of structurally and immunologically intact recombinantmycobacterial proteins from fast-growing mycobacterial hosts.

[0047] The ability to produce recombinant products in a form thatclosely resembles their native state is important in the study ofmicrobial antigens and enzymes. Recent studies have highlighted thesuperiority of recombinant protein purified from mycobacterial hostscompared to E. coli-derived products, as assessed by structural andimmunological analysis (Garbe et al., 1993; Roche et al., 1996; Triccaset al., 1996). Previously we have demonstrated that sera from leprosypatients would only recognize the M. leprae 35 kDa protein if theantigen was produced in a form that resembles the native protein, basedon the binding of conformational dependent mabs and FPLC size exclusionanalysis (Triccas et al., 1996). We reconfirm such a finding withprotein produced using the acetamidase promoter expression system (FIG.12). Furthermore, the addition of 6 histidine residues to the C-terminusof the recombinant protein does not appear to affect its conformation,as there is little difference in the recognition of leprosy sera byhistidine-tagged and nonhistidine-tagged 35 kDa protein (FIG. 12). Theefficient expression of the 6-histidine tag in mycobacteria and thesimple and effective purification of our model protein by Ni-NTAaffinity chromatography (FIG. 10) suggest that this versatilepurification system, used successfully in a number of eucaryotic andprocaryotic expression systems (Crowe et al., 1994), could be morewidely applied to mycobacterial proteins. Furthermore, the histidinepurification system overcomes the problems involved with antibodyaffinity chromatography used in a number of studies to purifyrecombinant mycobacterial proteins (Roche et al., 1996; Triccas et al.,1996), such as the unavailability of appropriate antibodies or thepresence of homologues capable of binding the antibody. Together, theseresults suggest an application for the pJAM2 expression vector in theproduction of native-like recombinant mycobacterial proteins that can beexploited to correctly analyze protein function and antigenicity.

[0048] The invention will be further clarified by the followingexamples, which are intended to be purely exemplary of the invention.

EXAMPLES

[0049] Bacteria, Media and Growth Conditions

[0050] The bacterial strains and plasmids used in this study are listedin FIG. 8. E. coli DH5a or BL21 (DE3) pLysS cultures were routinelygrown in Luria B medium (Difco) at 37° C. Mycobacterium cultures weregrown in Middlebrook 7H9 medium (Difco) supplemented with Tween 0.05%,glycerol (0.2%) and ADC (glucose, 0.2%; BSA fraction V, 0.5%; and NaCl,0.085%) at 37° C. When required, antibiotics were added at the followingconcentrations: ampicillin (100 μg/ml), kanamycin (20 μg/ml).

[0051] Human and Cattle Sera

[0052] Serum specimens from 20 individuals with pulmonary orextra-pulmonary tuberculosis (M. tuberculosis infected) were obtainedfrom the Bligny sanatorium (France). Six sera from M. bovis infectedhuman tuberculous patients and 24 sera from BCG-vaccinated patientssuffering from other pathologies were respectively obtained fromInstitut Pasteur, (Madagascar), and the Centre de Biologie Médicalespécialisée (CBMS) (Institut Pasteur, Paris). Sera from tuberculouscattle (M. bovis infected) were obtained from CNEVA, (Maison Alfort).

[0053] Subcloning Procedures

[0054] Restriction enzymes and T4 DNA ligase were purchased fromGibco/BRL, Boehringer Mannheim and New England Biolabs. All enzymes wereused in accordance with the manufacturer's recommendations. A 1-kbladder of DNA molecular mass markers was from Gibco/BRL. DNA fragmentsused in the cloning procedures were gel purified using the Geneclean IIkit (BIO 101 Inc., La Jolla, calif.). Cosmids and plasmids were isolatedby alkaline lysis (Sambrook et al., 1989). Bacterial strains weretransformed by electroporation using the Gene Pulser unit (Bio-RadLaboratories, Richmond, Calif.).

[0055] Southern Blot Analysis and Colony Hybridization

[0056] DNA-fragments for radiolabeling were separated on 0.7% agarosegels (Gibco BRL) in a Tris-borate-EDTA buffer system (Sambrook et al.,1989) and isolated from the gel by using Geneclean II (BIO 101).Radiolabeling was carried out with the random primed labeling kitMegaprime (Amersham) with 5 μCi of (α⁻³²P)dCTP, and unincorporated labelwas removed by passing through a Nick Column (Pharmacia). Southernblotting was carried out in 0.4 M NaOH with nylon membranes (Hybond-N+,Amersham) according to the Southern technique (Southern, 1975),prehybridization and hybridization was carried out as recommended by themanufacturer using RHB buffer (Amersham). Washing at 65° C. was asfollows: two washes with 2×SSPE (150 mM NaCl, 8.8 mM NaH₂PO₄, 1 mM EDTApH 7.4)-SDS 0.1% of 15 minutes each, one wash with 1×SSPE-SES 0.1% for10 minutes, two washes with 0.7×SSPE-SDS 0.1% of 15 minutes each.Autoradiographs were prepared by exposure with X-ray film (Kodak X-OMAT)at −80° C. overnight. Colony hybridization was carried out using nylonmembrane disc (Hybond-N+0.45 um, Amersham). E. coli colonies adsorbed onthe membranes were lysed in a (0.5M NaOH, 1.5M NaCl) solution, beforebeing placed for one minute in a microwave oven to fix the DNA.Hybridization and washes were described for the Southern blottinganalysis.

[0057] DNA Sequencing and Analysis

[0058] Sequences of double-stranded plasmid DNA were determined by thedideoxy-chain termination method (Sanger et al., 1977) using the Taq DyeDeoxy Terminator Cycle sequencing Kit (Applied Biosystems), on a GeneAmpPCR System 9600 (Perkin Elmer), and run on a DNA Analysis System-Model373 stretch (Applied Biosystems). The sequence was assembled andprocessed using DNA strider (CEA, France) and the University ofWisconsin Genetics Computer Group *UWGCG) packages. The BLAST algorithm(Altschul et al., 1990) was used to search protein data bases forsimilarity.

[0059] Expression and Purification of the DES Protein in E. coli

[0060] A 1043 bp NdeI-BamHI fragment of the des gene was amplified byPCR using nucleotides JD8: (5′-CGGCATATGTCAGCCAAGCTGACCGACCTGCAG-3′)(SEQ ID NO:1), and JD9: (5′CCGGGATCCCGCGCTCGCCGCTCTGCATCGTCG-3′)(SEQ IDNO:2), and cloned into the NdeI-BamHI sites of pET14b (Novagen) togenerate pET-des. PCR amplifications were carried out in a DNA thermalCycler (Perkin Elmer), using Taq polymerase (Cetus) according to themanufacturer's recommendations. PCR consisted of one cycle ofdenaturation (95° C., 6 min) followed by 25 cycles of amplificationconsisting of denaturation (95° C., 1 min), annealing (57° C., 1 min),and primer extension (72° C., 1 min). In the pET-des vector, theexpression of the des gene is under control of the T7 bacteriophagepromoter and the DES antigen is expressed as a fusion protein containingsix histidine residues. Expression of the des gene was induced byaddition of 0.4 mM IPTG in the culture medium. The DES protein waspurified by using a nickel-chelate affinity resin according to therecommendations of the supplier (Qiagen, Chatsworth, Calif.)

[0061] SDS-PAGE and Immunoblotting

[0062] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) was carried out as described by (Laemmli, 1970). For Westernblotting experiments (immunoblotting), approximately 10 μg of DESpurified protein were run on a SDS-polyacrylamide gel and transferred onnitrocellulose membranes (Hybond C extra, Amersham) using a Bio-Rad minitransblot apparatus according to the recommendations of the manufacturer(Bio-Rad Laboratories, Richmond, Calif). Transfer yield was visualizedby transient staining with Ponceau Rouge. The membrane were incubatedwith human patient or cattle sera diluted {fraction (1/200)} at 37° C.for 1 hour and with a goat anti-human (Promega) or rabbit anti-cattle(Biosys) IgG alkaline phosphatase-conjugated secondary antibody diluted{fraction (1/2500)}′ for 30 minutes at 37° C. The color reaction wasperformed by addition of 5-bromo-4-chloro-3-indolylphosphate (0.165mg/ml) and toluidinum nitroblue tetrazolium (0.33 mg/ml) as substrates.

[0063] ELISA

[0064] The human or cattle sera were tested for antibodies against DESby enzyme-linked immunosorbent assay (ELISA). The 96-well micro-titertrays (Nunc, Rochester, N.Y.) were coated with 0.1 μg (per well) ofpurified DES protein in guanidine hydrochloride buffer A (6 M guanidinehydrochloride, 0.1 M NaH₂PO₄, 0.01 M Tris, pH 8) (1h at 37° C. and 16hat 4 C). After three washes, wells were saturated with bovine serumalbumin 3% in phosphate buffered saline (PBS) for 30 min. at roomtemperature. After three washes, sera diluted from {fraction (1/50)}° to{fraction (1/3200)}° in buffer (PBS, 0.1% Tween 20, 1% bovine serumalbumin) were added to the wells for 2h at 37° C. After three washes,the wells were treated with goat anti-human IgG-alkaline phosphataseconjugate (Promega, Madison, Wis.) diluted {fraction (1/4000)}° for 1hat 37° C. Then, 4 mg of p-nitrophenylphospate per ml were added assubstrate. After 20 minutes of incubation at 37° C., the plates wereread photometrically at an optical density of 405 nm in micro-ELISAAutoreader (Dynatech, Mames la Coquette, France).

[0065] Statistics

[0066] Antibody responses of the different sera tested were compared byusing the Student t test. P>0.05 was considered nonsignificant.

[0067] Nucleotide Sequence and Accession Number

[0068] The nucleotide sequences of des has been deposited in the GenomeSequence Data Base (GSDB) under the accession number U49839.

[0069] Cloning of the des Gene

[0070] The construction of a fusion library of M. tuberculosis genomicDNA to the phoA gene and its expression in M. smegmatis, described by(Lim et al., 1995), led to the isolation of several PhoA⁺ clones.pExp421 is the plasmid harbored by one of the PhoA⁺ clones selected fromthis library. Detection of enzymatically active alkaline phosphataseindicated that the pExp421 insert contains functional expression andexportation signals. Restriction analysis showed that pExp421 carries a1.1 kb insert. Partial determination of its sequence identified a 577 bpORF, named des, fused in frame to the phoA gene and presenting twomotifs, of 9 and 14 amino acids, conserved with solublestearoyl-acyl-carrier protein desaturases (Lim et al., 1995).

[0071] To isolate the full-length des gene, the M. tuberculosis H37RvpYUB18 genomic cosmid library (Jacobs et al., 1991), was screened bycolony hybridization with the 1.1 kb probe (probe A, see FIG. 1). Twohybridizing cosmids named C₃ and C₄ were selected for further isolationof the gene. C₃ and C₄ were cut with several restriction enzymes andsubjected to Southern blot analysis using the 1.1 kb fragment as aprobe.

[0072] The EcoRV restriction profile revealed a single hybridizingfragment of 4.5 kb which was subcloned into pBluescript KS⁻ (Stratagene,La Jolla, Calif.) to-give plasmid pDS-des.

[0073] Characterization of the des Gene

[0074] The DNA sequence of the full des ORF was determined (FIG. 7). Thedes gene was shown to cover a 1017 bp region, encoding a 339 amino acidprotein with a calculated molecular mass of 37 kDa. The ORF starts witha potential ATG start codon at position 549, and ends with a TAG stopcodon at position 1565. There is a potential Shine-Delgarno motif(GGAGG) at position −8 upstream of the ATG. The G+C content of the ORF(62%) is consistent with the global GC content observed in themycobacterial genome. The nucleotide and deduced amino acid sequences ofthe des gene were compared to sequences in databases. They showed veryhigh homologies to the M. leprae aadx gene located on cosmid B2266,deposited in GenBank as part of the M. leprae genome sequencing project(GenBank accession number n°. U15182). Within the coding region, the DNAsequences were 79% identical while the encoded proteins were 80%identical (88% including conserved residues). The des gene also scoredsignificantly against soluble stearoyl-ACP desaturases: 44% identity atthe nucleotide level, 30% identity (51% including conserved residues) atthe amino acid level, to the Oryza saliva stearoyl-ACP desaturase(accession n°. D38753).

[0075] Although the detection of phoA enzymatic activity in the M.smegmatis clone harboring the pEXp421 suggests the DES protein isexported, no structural similarities were found between the DES proteinN terminal amino acids and signal sequences of bacterial exportedproteins (Izard & Kendall, 1994).

[0076] As in the M. leprae genome, a second ORF presenting highhomologies of the M. leprae putative NtrB gene (cosmid B2266), islocated downstream of the des gene in M. tuberculosis. Interestingly,the two ORF, des and Ntrb, are separated in M. tuberculosis by twodirect repeats of 66 nucleotides overlapping on 9 nucleotides (FIG. 2).

[0077] The DES Protein Presents the Conserved Amino Acid Motifs of theClass II diiron-oxo Proteins

[0078] Further analysis of the amino acid sequence of the DES proteinrevealed the presence of conserved motifs found only in class IIdiiron-oxo proteins (Fox et al. 1994) (FIG. 3). These proteins areoxo-bridged diiron clusters (Fe—O—Fe) containing proteins. They possessin their secondary structure 4 alpha helices involved in theprotein-derived cluster ligands. As revealed by X-ray structure studies,in these proteins, the diiron axis is oriented parallel to the long axisof the four helix bundle with ligands arising from four noncontiguoushelices, B, C, E and F. M. tuberculosis DES protein appears to have thesame active site residues as the class II diiron-oxo enzymes. Thisincludes Glu and His residues (E₁₀₇ and H₁₁₀ in helix C, E₁₆₇ in helix Eand E₁₉₇ and H₂₀₀ in helix F) that are ligands to the iron atoms, Asp,Glu and Arg residues (E106 and R₁₀₉ in helix C, D₁₉₆ in helix F) thatare involved in a hydrogen-bonding network to the cluster and, Ile andThr residues that may be part of the O₂-binding site (T₁₇₀ in helix E,I₁₉₃ in helix F). Thus, the M. tuberculosis DES protein contains in itsprimary sequence a conserved EEXXH (SEQ ID NO:3) motif and a conservedDEXXH (SEQ ID NO:4) motif, where X represents any amino acid. Theconserved motifs are separated by 85 amino acids.

[0079] The class II diiron-oxo protein family contains up to dateribonucleotide reductases, hydrocarbon hydroxylases (methanemono-oxygenase, toluene-4-mono-oxygenase and phenol hydroxylase) andsoluble-ACP desaturases. On the overall sequence alignment the DESprotein presents higher homology to soluble stearoyl-ACP desaturasesthan to ribonucleotide reductases or bacterial hydroxylases. Thepercentage identity at the amino acid level of the DES protein was saidto be 30% with the Oryza sativa stearoyl-ACP desaturases, whereas it isonly 17% with the Methylococcus capsulatus methane mono-oxygenase(accession n°. M60276) and 17.7% with the Epstein Barr ribonucleotidereductase (accession n°. V01555). Homologies to the soluble Δ9desaturases mostly concern the amino acids located within the activesite in helices C, E, and F (FIG. 3).

[0080] The method according to the invention can be carried out for thescreening and selection of molecules interacting with the enzymaticactivity of DES protein, for example, for acyl-ACP desaturase normallyproduced by higher plants.

[0081] The DES Protein Shares Structural Features with the PlantAcyl-ACP Desaturases

[0082] The three-dimensional structure of the DES protein was modeledbased on homology with the Ricinus communis Δ9 stearoyl-ACP desaturase.The structure of this plant desaturase was determined by proteincrystallography to 2.4 Å resolution (Lindqvist et al., 1996). The modelobtained has no Ramachandran outliers, has an excellent stereochemistryfor both main chain and side chains and has no bad contacts. 302residues out of the 337 total residues of the M. tuberculosis enzymecould be built based on the template's structure and aligned with anr.m.s. of 0.34 Å for their Ca atoms.

[0083] These 3.02 DES residues share 26% sequence identity with theresidues of plant Δ9 stearoyl-ACP desaturase. Thus, the structures ofthese 302 residues in the model represent a good approximation of theirtrue structure.

[0084] The plant Δ9 stearoyl-ACP desaturase and DES protein share almostcomplete sequence identity in the areas encoding the four helices, whichinclude the ligands for the bi-nuclear iron center, as well as in thesurrounding areas and in the area around the catalytic site. Therefore,one can be confident with the structure of the residues located withinthese areas that share substantial amino acid identity. (FIGS. 3a and 3b).

[0085] These areas include the part of the fatty acid binding site whichis close to the active site. From the structure of the Δ9 stearoyl-ACPdesaturase it was concluded that the fatty acid part of the substrate iscompletely buried in the enzyme, in a deep hydrophobic channel,positioning the site of desaturation between carbon 9 and 10 in the areaof the active site close to the binuclear iron center. (Lindqvist etal., 1996). The shape of the channel forces the substrate to bind in aconfirmation close to the product's cis-configuration. From amino acidsequence comparisons of plant desaturases it was further concluded thatthe size of the amino acid side chains at the bottom of this channeldetermines the chain length beyond the point of double bond insertionthat can be accepted by the various plant enzymes. (Cahoon et al.,1997). In the DES protein, the active site is completely conserved,suggesting that DES is evolutionarily related to the plant desaturases.If DES catalyzes a desaturation reaction, judging from the conservedshape of the substrate's pocket, the product of the enzymatic reactionwould have a cis-configuration around the introduced double bond.

[0086] Inspection of the bottom of the substrate channel in the model ofthe DES protein shows that the exchange of threonine T181 in the plantΔ9 stearoyl-ACP desaturase for the bulkier glutamine in DES (Q145) hasshortened the pocket significantly. This implies that the substrate inDES would have a maximum of seven carbons beyond the point of doublebond insertion as compared to nine carbons in the plant stearoyl-ACPdesaturase. Also, the replacement of methionine M114 in the plant enzymeby a negatively charged glutamic acid in DES (E85) could indicate thatthe substrate for the Des protein carries a polar or even positivelycharged group that can interact with this sidechain. Alternatively, thepolarity could make it difficult for hydrophobic fatty acid tails toreach the bottom of the already shorter cavity, thereby further limitingthe number of possible carbons beyond the point of double bond insertion(e.g., to five carbons). Other amino-acid substitutions in the bindingcleft do not affect the nature, shape and size of the substrate'sbinding cavity.

[0087] The electrostatic potential surface of the Δ9 stearoyl-ACPdesaturase and of the DES protein around the entrance of the substrate'sbinding channel are very different. This difference indicates that theDES protein and the plant Δ9 stearoyl-ACP desaturase may requiredifferent associated cofactors for activity and, in particular,different forms of fatty acid substrates.

[0088] Distribution of the des Gene in Other Mycobacterial Species

[0089] The presence of the des gene in PstI-digested chromosomal DNAfrom various mycobacterial strains was analyzed by Southern blotting(FIG. 4). The probe used (probe B) is a PCR amplification productcorresponding to nucleotides 572 to 1589 (see FIG. 1). The probehybridized on all mycobacterial genomic DNA tested. Strong signals weredetected in M. tuberculosis, M. bovis, M. bovis BCG, M. Africanum and M.avium. Weaker signals were visible in M. microti, M. xenopi, M.fortuitum and M. smegmatis. Thus, the des gene seems to be present insingle copy at least in the slow growing M. tuberculosis, M. bovis, M.bovis BCG, M. africanum, M. avium and M. xenopi as well as in the fastgrowing M. smegmatis.

[0090] Expression of the des Gene in E. coli

[0091] In order to over express the DES protein, the des gene wassubcloned into the bacteriophage T7 promoter-based expression vectorpET14b (Novagen). A PCR amplification product of the des gene (seematerial and methods) was cloned into the NdeI-BamHI sites of thevector, leading to the plasmid pET-des. Upon IPTG induction of E. ColiBL21 DE3 pLysS cells harboring the plasmid pET-des, a protein of about40 kDa was overproduced. The 40 kDa size of the overproduced proteincorresponds with the molecular mass calculated from the deducedpolypeptide. As shown in FIG. 5, the great majority of the overproducedDES protein is present in the insoluble matter of E. coli cells. Thisprobably results from the precipitation of the over-concentrated proteinin E. coli cytoplasm resulting in the formation of inclusion bodies. Tobe able to dissolve the protein, the purification was carried out usinga nickel chelate affinity resin under denaturing conditions in guanidinehydrochloride buffers. Among all the conditions tested (pH, detergents,etc.), the only condition in which the protein could be eluted withoutprecipitating in the column and remain soluble, was in a buffercontaining 6 M guanidine hydrochloride.

[0092] Immunogenicity of the DES Protein After Infection

[0093] Twenty serum samples from M. tuberculosis infected human patients(4 with extra-pulmonary tuberculosis, 15 with pulmonary tuberculosis and1 with both forms of the disease), 6 sera from M. bovis infected humanpatients and 4 sera from M. bovis infected cattle were tested eitherpooled or taken individually in immunoblot experiments to determine thefrequency of recognition of the purified DES protein by antibodies frominfected humans or cattle. 20 out of the 20 sera from the M.tuberculosis infected human patients and 6 out of the 6 sera from the M.bovis infected human patients recognized the recombinant antigen asshown by the reaction with the 37 kDa band, (FIG. 9). Furthermore, apool of sera from human lepromatous leprosy patients also reactedagainst the DES antigen.

[0094] In contrast, the pool of serum specimens from M. bovis infectedcattle did not recognize the DES protein. These results indicate thatthe DES protein is highly immunogenic in tuberculosis human patients.Both pulmonary and extra-pulmonary tuberculosis patients recognize theantigen.

[0095] Magnitude of Human Patients' Antibody Responses

[0096] An enzyme-linked immunosorbent assay (ELISA) was used to comparethe sensitivity of the different serum samples from 20 tuberculosispatients (15 infected by M. tuberculosis and 5 infected by M. Bovis) tothe DES antigen. This technique was also carried out to compare thesensitivity of the antibody response to DES of the 20 tuberculosispatients to the antibody response of 24 patients (BCG-vaccinated)suffering from other pathologies. As shown in FIG. 6, patients sufferingfrom pathologies other than tuberculosis, react at low level to the DESantigen (average OD₄₀₅=0.17 for a serum dilution {fraction (1/100)}⁴)The average antibody response from the tuberculosis patients infected byM. tuberculosis or M. bovis against the same antigen is much moresensitive (OD₄₀₅=0.32 and OD₄₀₅=0.36 respectively, for a serum dilution{fraction (1/100)}⁴) This difference in the sensitivity of theimmunological response is statistically highly significant at everydilution from {fraction (1/50)}^(a) to {fraction (1/3200)}^(a) as shownby a Student I₉₃ test (I₉₅=5.18, 6.57, 6.16, 5.79, 4.43, 2.53 and 1.95,at sera dilutions {fraction (1/50)}^(a), {fraction (1/100)}^(a),{fraction (1/200)}⁴, {fraction (1/400)}^(a), {fraction (1/800)}^(a),{fraction (1/600)}^(a) and {fraction (1/3200)}^(a), respectively). Nodifferences in the sensitivity of the antibody response was noticedbetween patients suffering from pulmonary or extra-pulmonarytuberculosis.

[0097] Allelic Exchange of des Gene

[0098] We constructed an inactivated copy of the des gene by insertinginto the XhoI site of the ApaI/SacI restriction fragment carrying thedes gene (Jackson et al., 1997), a kanamycin (Km) resistance cassette.This (des:Krn) construct was then inserted, along with the XylE gene,which encodes the Pseudomonas catechol dioxygenase conferring uponmycobacteria a yellow color when sprayed with catechol (Pelicic et al.,1997), into the pJQ200 plasmid, a pbluescript-derived E. coli vectorcarrying the sacB gene. The resulting vector was called pJQdKX.

[0099] In a first experiment, we transformed M. bovis BCG with pJQdKXand tried to select mutants resulting from allelic exchange eventsinside the des locus by using a two step procedure such as the onedescribed by (Pelicic et al., 1996). In the first step, we selected, onkanamycin-containing medium, a transformant that has integrated thewhole vector inside its chromosome by a single crossing-over within thedes locus. In the second step, using the counter-selection properties ofthe sacB gene, we selected bacteria that have undergone a secondintrachromosomal crossing-over, resulting in the replacement of the wildtype copy of the des gene by its inactivated copy (des:Km), i.e.,allelic exchange mutants.

[0100] Although at the first step of the procedure, 100% of thetransformants resulted from the integration of the pJQdKX vector by asingle homologous recombination event, no allelic exchange mutants wereobtained after the second selection step. 99.53% of the (Km, Sucrose)resistant colonies obtained at the end of the selection procedure wereXylE+, indicating that they still carried the vector in their chromosomeand probably also carried mutations in the sacB gene resulting in theirsucrose-resistant phenotype. The 0.47% XylE− remaining colonies possiblycarried mutations in both the sacB and the XylE genes since geneticanalysis (genomic hybridization, PCR) indicated they were notdes-allelic exchange mutants. This result suggests that the des genemight be essential to M. bovis BCG.

[0101] In order to investigate this hypothesis, we performed a secondexperiment in which we inserted, using an integrative vector pAV6950(Moniz-Pereira et al., 1995), a second wild type copy of the des gene(carried on a ApaI-SacI restriction fragment; see above) in thechromosome of a M. bovis BCG transformant resulting from the firstselection step described above. The resulting M. bovis BCG thuscontained two wild type copies of the des gene in addition to the(des:Km) copy carried by the inserted pJQdKX vector. When the secondselection step was applied on a culture of this bacteria, 34% of the(Km-sucrose)-resistant colonies obtained were XylE−. Genetic analysis ofthese candidates revealed that all of them corresponded to allelicexchange mutants. The other 66% (Km-sucrose)-resistant and XylE+colonies probably carried mutations in the sacB gene.

[0102] Construction of the Acetamidase Promoter Expression Vector pJAM2

[0103] The acetamidase promoter region was amplified from plasmid pAMI1,which contains the M. smegmatis NCTC 9449 inducible acetamidase gene andupstream region (Mahenthiralingam et al., 1993), by use of primers HIS5:(CACGGTACCAAGCTTTCTAGCAGA) (SEQ ID NO:38), and HIS7:(GTCAGTGGTGGTGGTGGTGGTGTCTAGAAGTACTGGATCCGAAAACTACCTCG) (SEQ ID NO:39)-.The resulting 1.6 kb fragment was cloned into plasmid pJEM12 (Timm etal., 1994b) to give plasmid pJAM2 (FIG. 2A). The coding region of the M.leprae 35 kDa protein was amplified by primers JN8:(TAGCTGCAGGGATCCATGACGTCGGCT)(SEQ ID NO:40), and 35REV2(GTGTCTAGACTTGTACTCATG) (SEQ ID NO:41), and cloned into the BamHI/XbaIsites of pJAM2, yielding pJAM4. The gene encoding the M. tuberculosisDES antigen was amplified by primers JD17:(GGGTCTAGAACGACGGCTCATCGCCAGTTTGCC) (SEQ ID NO:42), and JD18:(CCCGGATCCATGTCAGCCAAGCTGACCGACCTG) (SEQ ID NO:43) and also cloned intothe BamHI/XbaI sites of pJAM2 to give plasmid pJAM21.

[0104] Expression and Purification of Recombinant Histidine-TaggedProtein from M. smegmatis

[0105] Plasmids pJAM4 and pJAM21 were introduced into M. smegmatismc²155 and kanamycin resistant colonies grown in M63 medium [7.6×10⁻²M(NH4)₂SO₄, 0.5M KH₂PO₄, 5.8×10⁻⁶M FeSO₄.7H₂O, pH 7] supplemented with 2%succinate (Sigma Chemical Co., St Louis, Mo.) for uninduced cultures or2% succinate and acetamide (Sigma) for induced cultures. Bacteria weregrown for 3 days, after which cells were harvested and sonicated 4 timesfor 1 minute. Sonicates were analyzed for expression of recombinantproteins by SDS-PAGE and immunoblotting with the anti-35 kDa monoclonalantibody (mAb) CS38 for the M. leprae 35 kDa protein (CS38 supplied byProfessor Patrick Brennan, Colorado State University, Colorado) or forthe M. tuberculosis DES antigen using an anti-DES murine-derivedpolyclonal antibody. For protein purification, the sonicates wereapplied to Ni-NTA resin (Qiagen Inc., CA) and bound protein was washedconsecutively with 5 mM, 20 mM and 40 mM imidazole (Sigma) in sonicationbuffer (1×PBS, 5% glycerol, 0.5 M NaCl and 5 mM MgCl₂). Protein waseluted with 200 mM imidazole in sonication buffer and dialyzed againstPBS. Nonhistidine-tagged M. leprae 35 kDa protein derived from M.smegmatis and the E. coli 35 kDa 6-histidine fusion protein werepurified as described previously (Triccas et al., 1996).

[0106] Protein Capture ELISA

[0107] ELISA plates were coated with the murine anti-M. leprae 35 kDamAb ML03 (50 mg/ml; supplied by Professor J. Ivanyi, HammersmithHospital, London, UK) and mycobacterial sonicates were added at aconcentration range of 0.1 mg/ml to 100 mg/ml. Plates were blocked with3% bovine serum albumin (BSA), washed, and anti-rabbit 35 kDa proteinpolyclonal antibody (1:1000) added. Binding was visualized usingalkaline phosphatase conjugated anti-rabbit IgG (Sigma) andn-nitro-phenyl-phosphate (NPP) (1 mg/ml). Protein amount was determinedby comparison with purified M. leprae 35 kDa protein concentrationstandards (Triccas et al., 1996).

[0108] Assessment of Protein Binding to Leprosy Sera by ELISA

[0109] Microtitre plates were coated with antigen (100 pg/ml to 100mg/ml) overnight at room temperature. Plates were washed, blocked with3% BSA, and pooled sera (diluted 1:100) added for 90 minutes at 37° C.Plates were washed, and alkaline phosphatase conjugated anti-human IgG(Sigma) added for 60 minutes at 37° C. Binding was visualized by theaddition of n-nitro-phenyl-phosphate (1 mg/ml) and absorbance wasmeasured at 405 nm.

[0110] Construction of the pJAM2 Vector and Utilization forOver-Expression of the Gene Encoding the 35 kDa Antigen of M. leprae inM. smegmatis

[0111] The promoter region of the gene encoding the acetamidase of M.smegmatis NCTC 9449 permits the inducible expression of the enzyme inthe presence of the substrate acetamide (Mahenthiralingam et al., 1993).In order to determine if the promoter could regulate the expression offoreign genes placed under its control, the vector pJAM2 was constructed(FIG. 2A). This plasmid contains 1.5 kb upstream of the acetamidasecoding region, DNA encoding the first 6 amino acids of the acetamidasegene, three restriction enzymes sites, and the coding region for 6histidine residues. Thus this vector should allow for the inducibleexpression of foreign genes cloned within it, while also permittingsimple purification of the recombinant protein by virtue of thepolyhistidine tag. In order to validate the system, the coding region ofthe M. leprae 35 kDa protein was amplified and cloned into theBamHI/XbaI sites of pJAM2 to give plasmid pJAM4. This protein is a majorantigen of M. leprae and represents a promising candidate as aleprosy-specific diagnostic reagent (Triccas et al., 1996). PlasmidpJAM4 was introduced into M. smegmatis mc²155, and recombinant coloniesgrown in minimal media containing 2% succinate in the presence orabsence of 2% acetamide. Sonicates were prepared and proteins analyzedby SDS-PAGE. As shown in FIG. 10A, a prominent band was visible ataround 37 kDa in cells grown in acetamide plus succinate (lane 2), butabsent from cells grown in succinate alone (lane 1). This band reactedin immunoblotting with mAb CS38, which is raised against the native M.leprae 35 kDa protein (FIG. 10B, lane 2).

[0112] Quantifying Expression of Recombinant Protein in M. smegmatisUsing the pJAM2 Vector

[0113] In order to quantify the level at which the 35 kDa protein wasbeing produced by virtue of the acetamidase promoter in M.smegmatis/pJAM4, antigen-capture ELISA was employed. As shown in FIG.11, no protein was detected in M. smegmatis/pJAM4 grown in succinatealone. When the same strain was grown in the presence of acetamide, the35 kDa protein represented approximately 8.6% of the total bacterialsonicate. The strength of expression was highlighted through comparisonwith protein levels in M. smegmatis harboring plasmid pWL19 (Winter etal., 1995), where expression of the 35 kDa protein-gene is driven by theβ-lactamase promoter of Mycobacterium fortuitum, one of the strongestmycobacterial promoters characterized to date (Timm et al., 1994; Timmet al., 1994b). While M. smegmatis/pWL19 produced high levels of 35 kDaprotein, representing 7.1% of the bacterial sonicate, this was around17% less recombinant protein than that detected in M. smegmatis/pJAM4.

[0114] Purification of Histidine-Tagged Protein from Recombinant M.smegmatis

[0115] We next determined if the high-level expression by virtue of theM. smegmatis acetamidase promoter could allow efficient purification ofthe 35 kDa protein using the 6 histidine residues attached to itsC-terminus. This system has been successfully used in a number ofeucaryotic and procaryotic expression systems, and is favored due itssimple and reliable purification procedure, coupled with minimal effectsof the histidine tag on the target protein conformation, function, andimmunogenicity (Crowe et al., 1994). Although this system had not beenused in mycobacteria before, it seemed an ideal choice to allow thesimple and rapid purification of structurally and immunologically intactrecombinant mycobacterial proteins. Sonicates of M. smegmatis/pJAM4grown in the presence of acetamide were added to Ni-NTA resin (QiagenInc., CA), the column washed consecutively with varying amounts ofimidazole (5 mM, 20 mM and 40 mM) and protein eluted with 200 mMimidazole. This single-step procedure allowed 35 kDa protein ofpredominantly a single species to be purified (FIG. 10A, lane 3). Thepurified product reacted with the anti-M. leprae 35 kDa protein mAb CS38(FIG. 10B, lane 3). Therefore the strategy of Ni-NTA affinitychromatography by virtue of a polyhistidine tag can be utilized for theefficient purification of recombinant proteins from mycobacteria.

[0116] Analysis of the Effect of the Histidine Tag on RecombinantProtein Conformation and Immunogenicity

[0117] Previously it was demonstrated that recombinant forms of the M.leprae 35 kDa protein will only react with sera from leprosy patients ifthe protein is produced in a conformation that resembles that of thenative antigen (Triccas et al., 1996). This property allowed us to testthe effect, if any, of the histidine tag on the conformation of therecombinant 35 kDa protein. Three preparations of recombinant 35 kDaprotein were used: the histidine-tagged version purified in this study,a nonhistidine-tagged version purified from M. smegmatis, and an E. coli35 kDa 6-histidine fusion protein. The two latter proteins were purifiedas described previously (Triccas et al., 1996). The binding of pooledlepromatous leprosy sera to these three forms of the 35 kDa protein wereassessed by ELISA. The sera did not react with the E. coli form of the35 kDa protein (FIG. 12). By contrast, the 35 kDa-histidine fusionprotein purified from M. smegmatis/pJAM4 was strongly recognized by thesera. Furthermore, similar reactivity was exhibited towards the sameprotein purified from M. smegmatis containing no additional histidineresidues, suggesting that the addition of the histidine tag had noapparent effect on the conformation and indeed immunogenicity of therecombinant protein.

[0118] Induction and Over-Expression of the Gene Encoding the M.Tuberculosis DES Antigen Using the pJAM2 Expression System

[0119] To demonstrate that pJAM2 can be used for the induction andexpression of other genes placed within it, we cloned the gene encodingthe M. tuberculosis DES antigen into the BamHI/XbaI sites of the vector,to give pJAM21. The DES antigen is an immunodominant B-cell antigen withsignificant sequence similarity to plant acyl-acyl carrier proteindesaturases (Jackson et al, 1997). As assessed by immunoblot, noexpression of the DES gene was observed in M. smegmatis alone grown inthe presence or absence of acetamide (FIG. 13, lanes 1 and 2), or by M.smegmatis harboring pJAM21 (strain MYC1553) grown in the absence ofacetamide (FIG. 13, lanes 3 and 5). By contrast, the DES antigen wasreadily detected in sonicates of MYC1553 grown in the presence of 2%acetamide (FIG. 13, lanes 4 and 6). These results indicate thathigh-level induction of the des gene could be achieved by use of thepJAM2 expression system.

[0120] The references cited herein are listed on the following pages andare expressly incorporated by reference.

[0121] Other embodiments of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. It is intended that the specificationand examples be considered as exemplary only, with a true scope andspirit of the invention being indicated by the following claims.

BIBLIOGRAPHY

[0122] 1. Altschul, S. F., W. Gish, W. Miller, E. M. Myers, and D. J.Lipman. 1990. Basic local alignment search tool. Journal of MolecularBiology. 215:403-410.

[0123] 2. Anderson, A. B., and B. Brennan. 1994. Proteins and antigensof Mycobacterium tuberculosis, p. 307-332. In B. R. Bloom (ed.),Tuberculosis: Pathogenesis, Protection, and Control. ASM, Washington,D.C.

[0124] 3. Andersen, P. 1994. Effective vaccination of mice againstMycobacterium tuberculosis infection with a soluble mixture of secretedmycobacterial proteins. Infect. Immun. 62:2536-2544.

[0125] 4. Berthet, F. X., J. Rauzier, E. M. Lim, W. Philipp, B. Giequel,and D. Portnoi. 1995. Characterization of the M. tuberculosis erp geneencoding a potential cell surface protein with repetitive structures,Microbiology. 141:2123-2130.

[0126] 5. de Boer, H. A., Comstock, L. J. and Vasser, M. 1983. The tacpromoter: a functional hybrid derived from the trp and lac promoters.Proc. Natl. Acad. Sci. USA 80, 21-25.

[0127] 6. Braibant, M., L. D. Wit, P. Peirs, M. Kalai, J. Ooms, A.Drowart, K. Huygen, and J. Content. 1994. Structure of the Mycobacteriumtuberculosis antigen 88, a protein related to the Escherichia coli PstAperiplasmic phosphate permease subunit. Infection and Imununity.62:849-854.

[0128] 7. Cahoon, E. B., Lindqvist Y., Schneider, G., Shanklin, J. 1997.Redesign of soluble fatty acid desaturases from plants for alteredsubstrate specificity and double bond position. Proc. Nat'l. Acad. Sci.USA 94(10), pp. 4872-4877.

[0129] 8. Crowe, J., Dobeli, H., Gentz, E., Hochilu, E., Stuber, D. andHenco, K. 1994. 6×HIS-Ni-NTA chromatography as a superior technique inrecombinant protein expression/purification. Methods Mol. Biol. 31,371-387.*

[0130] 9. Fox, B. G., J. Shanklin, J. Ai, T. M. Loerh, and J.Sanders-Loerb. 1994. Resonance Raman evidence for an Fe—O—Fe center instearoyl-ACP desaturase. Primary sequence identity with other diiron-oxoproteins. Biochemistry. 33:12776-12786.

[0131] 10. Fulco, A. J., and K. Bloch. 1962. Cofactor requirements forfatty acid desaturation in Mycobacterium phlei. Biochim. Biophys. Acta.63:545-5-46.

[0132] 11. Fulco, A. J., and K. Bloch. 1964. Cofactor requirements forthe formation of Δ9 unsaturated fatty acids in Mycobacterium phlei. TheJournal of Biological Chemistry. 239-993-997.

[0133] 12. Garbe, T., Harris, D., Vordermeier, M., Lathigra, R., Ivanyi,J. and Young, D. 1993. Expression of the Mycobacterium tuberculosis19-kilodalton antigen in Mycobacterium smegmatis: immunological analysisand evidence of glycosylation. Infect. Immun. 61, 260-267.

[0134] 13. Gordon, S., Parish, T., Roberts, I. S. and Andrew, P. W.1994. The application of luciferase as a reporter of environmentalregulation of gene expression in mycobacteria. Lett. Appl. Microbiol.19, 336-340.

[0135] 14. Haslov, K., A. Andersen, S. Nagai, A. Gottschau, T. Sorensen,and P. Andersen. 1995. Guinea pig cellular immune responses to proteinssecreted by Mycobacterium tuberculosis. Infection and Immunity.63:804-810.

[0136] 15. Hatfull, G. F, 1993. Genetic transformation of mycobacteria.

[0137] Trends in microbiology. 1:310-314.

[0138] 16. Hermans, P. W. M., F. Abebe, V. I. O. Kuteyi, A. H. J. Kolk,J. E. R. Thole, and M. Harboe. 1995. Molecular and immunologicalcharacterization of the highly conserved antigen 84 from Mycobacteriumtuberculosis and Mycobacterium leprae. Infection and Immunity.63:954-960.

[0139] 17. Horburgh, C. R. 1991. Mycobacterium avium complex infectionsin the acquired immunodeficiency syndrome. New England, Journal ofMedicine, Vol. 34, pages 1332-1338.

[0140] 18. Izard, J. W., and D. A. Kendall. 1994. Signal peptides:exquisitely designed transport promoters. Molecular Microbiology.13:765-773.

[0141] 19. Jackson, M., Portnoï, D., Catheline, D., Dumail, L., Rauzier,J., Legrand, P. and Gicquel, B. 1997. Mycobacterium tuberculosis DESprotein: an immunodominant target for the humoral immune response oftuberculosis patients. Infect. Immun. 65, 2883-2889.

[0142] 20. Jacobs, W. R., G. V. Kalpana, J. D. Cirillo, L. Pascopella,S. B. Snapper, R. A. Udani, W. Jones, R. G. Barletta, and B. R. Bloom.1991. Genetic systems for mycobacteria. Methods Enzymol. 204:537-555.

[0143] 21. Kasbiwabara, Y., H. Nakagawa, G. Matsuki, and R. Sato. 1975.Effect of metal ions in the culture medium on the stearoyl-Coenzyme Adesaturase activity of Mycrobacterium phlei. J. Biochem. 78:803-810.

[0144] 22. Kashiwabara, Y., and R. Sato. 1973. Electron transfermechanism involved in stearoyl-coenzyme A desaturation by particulatefraction of Mycrobacterium phlei. J. Biochem. 74:405-413.

[0145] 23. Keegstra, K., and L. J. Olsen. 1989. Chloroplastic precursorsand their transport across the envelope membranes. Ann. Rev. PlantPhysiol. Plant Mol. Biol. 40:471-501.

[0146] 24. Laemmli, U. K. 1970. Cleavage of structural proteins duringthe assembly of the head of bacteriophage T4. Nature (London).227:680-685.

[0147] 25. Lee, B. Y., S. A. Hefta, and P. J. Brennan. 1992.Characterization of the major membrane protein of virulent Mycobacteriumtuberculosis. Infection and Immunity. 60:2066-2074.

[0148] 26. Legrand, P., and A. Bensadoun. 1991. Stearoyl-CoA desaturaseactivity in cultured rat hepatocytes. Biochimica et Biophysica Acia.1086:89-94.

[0149] 27. Lim, E. M., J. Rauzier, J, Timm, G. Torrea, A. Murray, B.

[0150] Gicquel, and D. Portnoi. 1995. Identification of Mycobacteriumtuberculosis DNA sequences encoding exported proteins by using phoA genefusions. Journal of Bacteriology. 177:59-65.

[0151] 28. Lindqvist, Y., Huang, W., Schneider, G., Shanklin, J. 1996.Crystal structure of delta9 stearoyl-acyl carrier protein desaturasefrom castor seed and its relationship to other di-iron proteins. EMBO.15(16):4081-92.

[0152] 29. Mahenthiralingam, E., Draper, P., Davis, E. O. and Colston,M. J. 1993. Cloning and sequencing of the gene which encodes the highlyinducible acetamidase of Mycobacterium smegmatis. J. Gen. Microbiol.139, 575-583.

[0153] 30. Pal, P. G., and M. A. Horwitz: 1992. Immunization withextracellular proteins of Mycobacterium tuberculosis inducescell-mediated immune responses and substantial protective immunity in aguinea pig model of pulmonary tuberculosis. Infection and Immunity.60:4781-4792.

[0154] 31. Parish, T., Mahenthiralingam, E., Draper, P., Davis, E. O.and Colston, M. J. 1997. Regulation of the inducible acetamidase gene ofMycobacterium smegmatis. Microbiology 143, 2267-2276.

[0155] 32. Parish, T. and Stocker, N. G. 1997b. Development and use of aconditional antisense mutagenesis system in mycobacteria. FEMSMicrobiol. Lett. 154, 151-157.

[0156] 33. Pelicic et al.: 1997. Efficient allelic exchange andtransposon mutagenesis in mycobacterium tuberculosis. Proc. Natl. Acad.Sci. USA, 94:10955-10960.

[0157] 34. Pelicic et al.: 1996. Generation of unmarked directedmutations in mycobacteria, using sucrose counter-selectable suicidevectors. Mol. Microbiol., 20:919-925.

[0158] 35. M. Picardeau and V. Vincent: 1995. Development of aspecies-specific probe for Mycobacterium xenopi Res. Microbiol.,46:237-263.

[0159] 36. Roche, P. W.; Winter, N., Triccas, J. A., Feng, C. andBritton, W. J. 1996. Expression of Mycobacterium tuberculosis MPT64 inrecombinant M. smegmatis: purification, immunogenicity and applicationto skin tests for tuberculosis. Clin. Exp. Immunol. 103, 226-232.

[0160] 37. Romain, F., A. Laqueyrerie, P. Militzer, P. Pescher, P.Chavarot, M.-Lagranderie, G. Auregan, M. Gheorghiu, and G. Marchal.1993. Identification of a Mycobacterium bovis BCG 45/47-kilodaltonantigen complex, an immunodominant target for antibody response afterimmunization with living bacteria. Infection and immunity. 61:742-750.

[0161] 38. Sakamoto, T., H. Wada, I. Nishida, M. Ohmori, and N. Murata.1994. Δ9 acyl lipid desaturases of cyanobacteria. J. Biol. Chem.269:25576-25580.

[0162] 39. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecularcloning—A laboratory manual. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.

[0163] 40. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNAsequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci.USA. 74:5463-5467.

[0164] 41. Shanklin, J., and C. Somerville. 1991.Stearoyl-acyl-carrier-protein desaturase from higher plants isstructurally unrelated to the animal and fungal homologs. Proceeding ofthe National Academy of Science of the United States of America.88:2510-2514.

[0165] 42. Shanklin, J., E. Whittle, and B. G. Fox. 1994. Eighthistidine residues art catalytically essential in a membrane-associatediron enzyme, stearoyl-CoA desaturase, and are conserved in alkanehydroxylase and xylene mono-oxygenase. Biochemistry. 33:12787-12794.

[0166] 43. Snapper, S. B., B. R. Bloom, and J. W. R. Jacobs. 1990.Molecular genetic approaches to mycobacterial investigation. p. 199-218.In J. McFadden (ed.), Molecular Biology of the Mycobacteria. SurreyUniversity Press, London.

[0167] 44. Sorensen, A. L., S. Nagai, G. Houen, P. Andersen, and A. B.

[0168] Andersen. 1995. Purification and characterization of alow-molecular-mass T-cell antigen secreted by Mycobacteriumtuberculosis. Infection and Immunity. 63:1710-1717.

[0169] 45. Southern, E. M. 1975. Detection of specific sequences amongDNA fragments separated by gel electrophoresis. J. Mol. Biol.98:503-517.

[0170] 46. Studier, W., A. H. Rosenberg, J. J. Dunn, and J. W.Dubendorff. 1990. Use of T7 RNA polymerase to direct expression ofcloned genes.

[0171] Methods in Enzymology. 185:60-89.

[0172] 47. Thole, J. E. R., and R. v. d. Zee. 1990. The 65 kDa antigen:molecular studies on a ubiquitous antigen., p. 37-66. In J. McFadden(ed.). Molecular Biology of the mycobacteria. Surrey University Press.London.

[0173] 48. Timm, J., Lim, E.M. and Gicquel, B. 1994b. Escherichiacoli-mycobacteria shuttle vectors for operon and gene fusions to lacZ:the pJEM series. J. Bacteriol. 176, 6749-6753.

[0174] 49. Timm, J., Perilli, M.G., Duez, C., Trias, J., Orefici, G.,Fattorini, L., Amicosante, G., Oratore, A., Joris, B., Frere, J. M.,Pugsley, A. P. and Gicquel, B. 1994. Transcription and expressionanalysis, using lacZ and phoA gene fusions, of Mycobacterium fortuitumb-lactamase genes cloned from a natural isolate and a high-levelb-lactamase producer. Mol. Microbiol. 12, 491-504.

[0175] 50. Triccas, J. A., Roche, P. W., Winter, N., Feng, C. G.,Butlin, C. R. and Britton, W. J. 1996. A 35 kDa protein is a majortarget of the human immune response to Mycobacterium leprae. Infect.Immun. 64: 5171-5177.

[0176] 51. Wheeler, P. R., and C. Ratledge. 1994. Metabolism ofMycobacterium tuberculosis, p. 353-385. In B. R. Bloom (ed.),Tuberculosis: Pathogenesis, Protection, and Control. ASM, Washington,D.C.

[0177] 52. Winter, N., Triccas, J. A., Rivoire, B., Pessolani, M. C. V.,Eiglmeier, K., Hunter, S. W., Brennan, P. J. and Britton, W. J. 1995.Characterization of the gene encoding the immunodominant 35 kDa proteinof Mycobacterium leprae. Mol. Microbiol. 16, 865-876.

[0178] 53. Young, D., T. Garbe, R. Lathigra, and C. Abou-Zeid. 1990.Protein antigens: structure, function and regulation, p. 1-35. In J.McFadden (ed.). Molecular biology of mycobacteria. Surrey universityPress, Laudon.

[0179] 54. Young, R. A., B. R. Bloom, C. M. Grossinsky, J. lvany, D.Thomas, and R. W. Davis. 1985. Dissection of the Mycobacteriumtuberculosis antigens using recombinant DNA. Proc. Natl. Acad. Sci.USA.82:2583-2587.

1 45 1 33 DNA Artificial Sequence Description of Artificial Sequenceprimer 1 cggcatatgt cagccaagct gaccgacctg cag 33 2 33 DNA ArtificialSequence Description of Artificial Sequence primer 2 ccgggatcccgcgctcgccg ctctgcatcg tcg 33 3 5 PRT Artificial Sequence Description ofArtificial Sequence motif 3 Glu Glu Xaa Xaa His 1 5 4 5 PRT ArtificialSequence Description of Artificial Sequence motif 4 Asp Glu Xaa Xaa His1 5 5 10 PRT Artificial Sequence Description of Artificial Sequencemotif 5 Asp Glu Xaa Xaa His Glu Glu Xaa Xaa His 1 5 10 6 52 PRT EpsteinBarr virus 6 Glu Phe Tyr Lys Phe Leu Phe Thr Phe Leu Ala Met Ala Glu LysLeu 1 5 10 15 Val Asn Phe Asn Ile Asp Glu Leu Val Thr Ser Phe Glu SerHis Asp 20 25 30 Ile Asp His Tyr Tyr Thr Glu Gln Lys Ala Met Glu Asn ValHis Gly 35 40 45 Glu Thr Tyr Ala 50 7 52 PRT E. coli 7 Ile Phe Ile SerAsn Leu Lys Tyr Gln Thr Leu Leu Asp Ser Ile Gln 1 5 10 15 Gly Arg SerPro Asn Val Ala Leu Leu Pro Leu Ile Ser Ile Pro Glu 20 25 30 Leu Glu ThrTrp Val Glu Thr Trp Ala Phe Ser Glu Thr Ile His Ser 35 40 45 Arg Ser TyrThr 50 8 52 PRT Methylcoccus capsulatus 8 Glu Thr Met Lys Val Val SerAsn Phe Leu Glu Val Gly Glu Tyr Asn 1 5 10 15 Ala Ile Ala Ala Thr GlyMet Leu Trp Asp Ser Ala Gln Ala Ala Glu 20 25 30 Gln Lys Asn Gly Tyr LeuAla Gln Val Leu Asp Glu Ile Arg His Thr 35 40 45 His Gln Cys Ala 50 9 52PRT Methylosinus trichosporium 9 Glu Thr Met Lys Val Ile Ser Asn Phe LeuGlu Val Gly Glu Tyr Asn 1 5 10 15 Ala Ile Ala Ala Ser Ala Met Leu TrpAsp Ser Ala Thr Ala Ala Glu 20 25 30 Gln Lys Asn Gly Tyr Leu Ala Gln ValLeu Asp Glu Ile Arg His Thr 35 40 45 His Gln Cys Ala 50 10 52 PRTPseudomonas sp. 10 Asn Ala Leu Lys Leu Phe Leu Thr Ala Val Ser Pro LeuGlu Tyr Gln 1 5 10 15 Ala Phe Gln Gly Phe Ser Arg Val Gly Arg Gln PheSer Gly Ala Gly 20 25 30 Ala Arg Val Ala Cys Gln Met Gln Ala Ile Asp GluLeu Arg His Val 35 40 45 Gln Thr Gln Val 50 11 52 PRT Pseudomonasmendocina 11 Ser Thr Leu Lys Ser His Tyr Gly Ala Ile Ala Val Gly Glu TyrAla 1 5 10 15 Ala Val Thr Gly Glu Gly Arg Met Ala Arg Phe Ser Lys AlaPro Gly 20 25 30 Asn Arg Asn Met Ala Thr Phe Gly Met Met Asp Glu Leu ArgHis Gly 35 40 45 Gln Leu Gln Leu 50 12 54 PRT Ricinus communis 12 LeuVal Gly Asp Met Ile Thr Glu Glu Ala Leu Pro Thr Tyr Gln Thr 1 5 10 15Met Leu Asn Thr Leu Asp Gly Val Arg Asp Glu Thr Gly Ala Ser Pro 20 25 30Thr Ser Trp Ala Ile Trp Thr Arg Ala Trp Thr Ala Glu Glu Asn Arg 35 40 45His Gly Asp Leu Leu Asn 50 13 54 PRT Cucumis sativus 13 Leu Val Gly AspMet Ile Thr Glu Glu Ala Leu Pro Thr Tyr Gln Thr 1 5 10 15 Met Leu AsnThr Leu Asp Gly Val Arg Asp Glu Thr Gly Ala Ser Pro 20 25 30 Thr Pro TrpAla Ile Trp Thr Arg Ala Trp Thr Ala Glu Glu Asn Arg 35 40 45 His Gly AspLeu Leu Asn 50 14 54 PRT Carthamus tinctorius 14 Leu Val Gly Asp Met IleThr Glu Glu Ala Leu Pro Thr Tyr Gln Thr 1 5 10 15 Met Leu Asn Thr LeuAsp Gly Val Arg Asp Glu Thr Gly Ala Ser Leu 20 25 30 Thr Pro Trp Ala ValTrp Thr Arg Ala Trp Thr Ala Glu Glu Asn Arg 35 40 45 His Gly Asp Leu LeuHis 50 15 54 PRT Spinacia oleracea 15 Leu Val Gly Asp Met Ile Thr GluGlu Ala Leu Pro Thr Tyr Gln Thr 1 5 10 15 Met Leu Asn Thr Leu Asp GlyAla Lys Asp Glu Thr Gly Ala Ser Pro 20 25 30 Thr Ser Trp Ala Val Trp ThrArg Ala Trp Thr Ala Glu Glu Asn Arg 35 40 45 His Gly Asp Leu Leu Asn 5016 54 PRT Brassica rapa 16 Leu Val Gly Asp Met Ile Thr Glu Glu Ala LeuPro Thr Tyr Gln Thr 1 5 10 15 Met Leu Asn Thr Leu Asp Gly Val Arg AspGlu Thr Gly Ala Ser Pro 20 25 30 Thr Ser Trp Ala Ile Trp Thr Arg Ala TrpThr Ala Glu Glu Asn Arg 35 40 45 His Gly Asp Leu Leu Asn 50 17 54 PRTSolanum tuberosum 17 Leu Ile Gly Asp Met Ile Thr Glu Glu Ala Leu Pro ThrTyr Gln Thr 1 5 10 15 Met Ile Asn Thr Leu Asp Gly Val Arg Asp Glu ThrGly Ala Thr Val 20 25 30 Thr Pro Trp Ala Ile Trp Thr Arg Ala Trp Thr AlaGlu Glu Asn Arg 35 40 45 His Gly Asp Leu Leu Asn 50 18 54 PRT Linumusitatissimum 18 Leu Val Gly Asp Met Ile Thr Glu Glu Ala Leu Pro Thr TyrGln Thr 1 5 10 15 Met Leu Asn Thr Leu Asp Gly Val Arg Asp Glu Thr GlyAla Ser Leu 20 25 30 Thr Pro Trp Ala Ile Trp Thr Arg Ala Trp Thr Ala GluGlu Asn Arg 35 40 45 His Gly Asp Leu Leu Asn 50 19 54 PRT Coriandrumsativum 19 Leu Val Gly Asp Met Ile Thr Glu Glu Ala Leu Pro Thr Tyr MetSer 1 5 10 15 Met Leu Asn Arg Cys Asp Gly Ile Lys Asp Asp Thr Gly AlaGln Pro 20 25 30 Thr Ser Trp Ala Thr Trp Thr Arg Ala Trp Thr Ala Glu GluAsn Arg 35 40 45 His Gly Asp Leu Leu Asn 50 20 54 PRT Mycobacteriumtuberculosis 20 Ser Asp Val Ala Gln Val Ala Met Val Gln Asn Leu Val ThrGlu Asp 1 5 10 15 Asn Leu Pro Ser Tyr His Arg Glu Ile Ala Met Asn MetGly Met Asp 20 25 30 Gly Ala Trp Gly Gln Trp Val Asn Arg Trp Thr Ala GluGlu Asn Arg 35 40 45 His Gly Ile Ala Leu Arg 50 21 52 PRT Epstein Barrvirus 21 Glu Lys Ile Leu Val Phe Leu Leu Ile Glu Gly Ile Phe Phe Ile Ser1 5 10 15 Ser Phe Tyr Ser Ile Ala Leu Leu Arg Val Arg Gly Leu Met ProGly 20 25 30 Ile Cys Leu Ala Asn Asn Tyr Ile Ser Arg Asp Glu Leu Leu HisThr 35 40 45 Arg Ala Ala Ser 50 22 52 PRT E. coli 22 Leu Cys Leu Met SerVal Asn Ala Leu Glu Ala Ile Arg Phe Tyr Val 1 5 10 15 Ser Phe Ala CysSer Phe Ala Phe Ala Glu Arg Glu Leu Met Glu Gly 20 25 30 Asn Ala Lys IleIle Arg Leu Ile Ala Arg Asp Glu Ala Leu His Leu 35 40 45 Thr Gly Thr Gln50 23 52 PRT Methylcoccus capsulatus 23 Cys Ser Leu Asn Leu Gln Leu ValGly Glu Ala Cys Phe Thr Asn Pro 1 5 10 15 Leu Ile Val Ala Val Thr GluTrp Ala Ala Ala Asn Gly Asp Glu Ile 20 25 30 Thr Pro Thr Val Phe Leu SerIle Glu Thr Asp Glu Leu Arg His Met 35 40 45 Ala Asn Gly Tyr 50 24 52PRT Methylosinus trichosporium 24 Cys Ser Val Asn Leu Gln Leu Val GlyAsp Thr Cys Phe Thr Asn Pro 1 5 10 15 Leu Ile Val Ala Val Thr Glu TrpAla Ile Gly Asn Gly Asp Glu Ile 20 25 30 Thr Pro Thr Val Phe Leu Ser ValGlu Thr Asp Glu Leu Arg His Met 35 40 45 Ala Asn Gly Tyr 50 25 52 PRTPseudomonas sp. 25 Phe Leu Thr Ala Val Ser Phe Ser Phe Glu Tyr Val LeuThr Asn Leu 1 5 10 15 Leu Phe Val Pro Phe Met Ser Gly Ala Ala Tyr AsnGly Asp Met Ala 20 25 30 Thr Val Thr Phe Gly Phe Ser Ala Gln Ser Asp GluAla Arg His Met 35 40 45 Thr Leu Gly Leu 50 26 52 PRT Pseudomonasmendocina 26 Val Ala Ile Met Leu Thr Phe Ser Phe Glu Thr Gly Phe Thr AsnMet 1 5 10 15 Gln Phe Leu Gly Leu Ala Ala Asp Ala Ala Glu Ala Gly AspTyr Thr 20 25 30 Phe Ala Asn Leu Ile Ser Ser Ile Gln Thr Asp Glu Ser ArgHis Ala 35 40 45 Gln Gln Gly Gly 50 27 52 PRT Ricinus communis 27 TyrLeu Gly Phe Ile Tyr Thr Ser Phe Gln Glu Arg Ala Thr Phe Ile 1 5 10 15Ser His Gly Asn Thr Ala Arg Gln Ala Lys Glu His Gly Asp Ile Lys 20 25 30Leu Ala Gln Ile Cys Gly Thr Ile Ala Ala Asp Glu Lys Arg His Glu 35 40 45Thr Ala Tyr Thr 50 28 52 PRT Cucumis sativus 28 Tyr Leu Gly Phe Ile TyrThr Ser Phe Gln Glu Arg Ala Thr Phe Ile 1 5 10 15 Ser His Gly Asn ThrAla Arg Leu Ala Lys Glu His Gly Asp Ile Lys 20 25 30 Leu Ala Gln Ile CysGly Thr Ile Thr Ala Asp Glu Lys Arg His Glu 35 40 45 Thr Ala Tyr Thr 5029 52 PRT Carthamus tinctorius 29 Tyr Leu Gly Phe Ile Tyr Thr Ser PheGln Glu Arg Ala Thr Phe Val 1 5 10 15 Ser His Gly Asn Thr Ala Arg HisAla Lys Asp His Gly Asp Val Lys 20 25 30 Leu Ala Gln Ile Cys Gly Thr IleAla Ser Asp Glu Lys Arg His Glu 35 40 45 Thr Ala Tyr Thr 50 30 52 PRTSpinacia oleracea 30 Tyr Leu Gly Phe Val Tyr Thr Ser Phe Gln Glu Arg AlaThr Phe Val 1 5 10 15 Ser His Gly Asn Ser Ala Arg Leu Ala Lys Glu HisGly Asp Leu Lys 20 25 30 Met Ala Gln Ile Cys Gly Ile Ile Ala Ser Asp GluLys Arg His Glu 35 40 45 Thr Ala Tyr Thr 50 31 52 PRT Brassica rapa 31Tyr Leu Gly Phe Ile Tyr Thr Ser Phe Gln Glu Arg Ala Thr Phe Ile 1 5 1015 Ser His Gly Asn Thr Ala Arg Gln Ala Lys Glu His Gly Asp Leu Lys 20 2530 Leu Ala Gln Ile Cys Gly Thr Ile Ala Ala Asp Glu Lys Arg His Glu 35 4045 Thr Ala Tyr Thr 50 32 52 PRT Solanum tuberosum 32 Tyr Leu Gly Phe ValTyr Thr Ser Leu Arg Lys Gly Val Thr Phe Val 1 5 10 15 Ser His Gly AsnThr Ala Arg Leu Ala Lys Glu His Gly Asp Met Lys 20 25 30 Leu Ala Gln IleCys Gly Ser Ile Ala Ala Asp Glu Lys Arg His Glu 35 40 45 Thr Ala Tyr Thr50 33 52 PRT Linum usitatissimum 33 Tyr Leu Gly Phe Ile Tyr Thr Ser PheGln Glu Arg Ala Thr Phe Ile 1 5 10 15 Ser His Gly Asn Thr Ala Arg LeuAla Lys Asp His Gly Asp Met Lys 20 25 30 Leu Ala Gln Ile Cys Gly Ile IleAla Ala Asp Glu Lys Arg His Glu 35 40 45 Thr Ala Tyr Thr 50 34 52 PRTCoriandrum sativum 34 Tyr Met Gly Phe Ile Tyr Thr Ser Phe Gln Glu ArgAla Thr Phe Ile 1 5 10 15 Ser His Ala Asn Thr Ala Lys Leu Ala Gln HisTyr Gly Asp Lys Asn 20 25 30 Leu Ala Gln Val Cys Gly Asn Ile Ala Ser AspGlu Lys Arg His Ala 35 40 45 Thr Ala Tyr Thr 50 35 49 PRT Mycobacteriumtuberculosis 35 Thr Asp Ser Val Leu Tyr Val Ser Phe Gln Glu Leu Ala ThrArg Ile 1 5 10 15 Ser His Arg Asn Thr Gly Lys Ala Cys Asn Asp Pro ValAla Asp Gln 20 25 30 Leu Met Ala Lys Ile Ser Ala Asp Glu Asn Leu His MetIle Phe Tyr 35 40 45 Arg 36 1600 DNA Mycobacterium tuberculosis CDS(549)..(1562) 36 gatcatcatc ggccggctgc cgcgcagggc gccgacaccg gcgagtgcgggcgcgaggat 60 cggcccccac cagttcggca gctgcgtgtc gatgcgctcc acaatcccgggaaacagctc 120 gaccattacc tcctcaatat gagcctcgaa aaacttgccg ctgtgcgcggcgtcgtggtg 180 agcgcacaca acaactgtta gctgaccagc aggatcggcg ctcttaccggtctgttcacc 240 gcatatctga acggacggct gggagccacc cgcaagcaat tcatcgactactgcgtcaac 300 atgttgctca gcaccgccgc cacctacgca ccgcaccgcg agcggggagaatccgaacac 360 tccatcccag ccgggccgca caactgagga cgactggggt tcaccccacgcggccaccgg 420 cgcccgccga tgccagcatc ctgcccgctg ctggcagctc aacatgccgcgcgaagccca 480 aacttgatgc taccgagaga cacagatata ttgactgcaa ccattagacacagataactg 540 gaggcgcc atg tca gcc aag ctg acc gac ctg cag ctg ctg cacgaa ctt 590 Met Ser Ala Lys Leu Thr Asp Leu Gln Leu Leu His Glu Leu 1 510 gaa ccg gtc gtc gag aag tac ctg aac cgg cac ctg agc atg cac aag 638Glu Pro Val Val Glu Lys Tyr Leu Asn Arg His Leu Ser Met His Lys 15 20 2530 ccc tgg aac ccg cac gac tac atc ccg tgg tcg gac ggg aag aac tac 686Pro Trp Asn Pro His Asp Tyr Ile Pro Trp Ser Asp Gly Lys Asn Tyr 35 40 45tac gcg ctc ggc ggg cag gat tgg gac ccc gac cag agc aag ctt tct 734 TyrAla Leu Gly Gly Gln Asp Trp Asp Pro Asp Gln Ser Lys Leu Ser 50 55 60 gatgtc gcc cag gtg gcg atg gtg cag aac ctg gtc acc gag gac aac 782 Asp ValAla Gln Val Ala Met Val Gln Asn Leu Val Thr Glu Asp Asn 65 70 75 ctg ccgtcg tat cac cgc gag atc gcg atg aac atg ggc atg gac ggc 830 Leu Pro SerTyr His Arg Glu Ile Ala Met Asn Met Gly Met Asp Gly 80 85 90 gcg tgg gggcag tgg gtc aac cgt tgg acc gcc gag gag aat cgg cac 878 Ala Trp Gly GlnTrp Val Asn Arg Trp Thr Ala Glu Glu Asn Arg His 95 100 105 110 ggc atcgcg ctg cgc gac tac ctg gtg gtg acc cga tcg gtc gac cct 926 Gly Ile AlaLeu Arg Asp Tyr Leu Val Val Thr Arg Ser Val Asp Pro 115 120 125 gtc gagttg gag aaa ctt cgc ctc gag gta gtc aac cgg ggc ttc agc 974 Val Glu LeuGlu Lys Leu Arg Leu Glu Val Val Asn Arg Gly Phe Ser 130 135 140 cca ggccaa aac cac cag ggc cac tat ttc gcg gag agc ctc acc gac 1022 Pro Gly GlnAsn His Gln Gly His Tyr Phe Ala Glu Ser Leu Thr Asp 145 150 155 tcc gtcctc tat gtc agt ttc cag gaa ctg gca acc cgg att tcg cac 1070 Ser Val LeuTyr Val Ser Phe Gln Glu Leu Ala Thr Arg Ile Ser His 160 165 170 cgc aatacc ggc aag gca tgt aac gac ccc gtc gcc gac cag ctc atg 1118 Arg Asn ThrGly Lys Ala Cys Asn Asp Pro Val Ala Asp Gln Leu Met 175 180 185 190 gccaag atc tcg gca gac gag aat ctg cac atg atc ttc tac cgc gac 1166 Ala LysIle Ser Ala Asp Glu Asn Leu His Met Ile Phe Tyr Arg Asp 195 200 205 gtcagc gag gcc gcg ttc gac ctc gtg ccc aac cag gcc atg aag tcg 1214 Val SerGlu Ala Ala Phe Asp Leu Val Pro Asn Gln Ala Met Lys Ser 210 215 220 ctgcac ctg att ttg agc cac ttc cag atg ccc ggc ttc caa gta ccc 1262 Leu HisLeu Ile Leu Ser His Phe Gln Met Pro Gly Phe Gln Val Pro 225 230 235 gagttc cgg cgc aaa gcc gtg gtc atc gcc gtc ggg ggt gtc tac gac 1310 Glu PheArg Arg Lys Ala Val Val Ile Ala Val Gly Gly Val Tyr Asp 240 245 250 ccgcgc atc cac ctc gac gaa gtc gtc atg ccg gta ctg aag aaa tgg 1358 Pro ArgIle His Leu Asp Glu Val Val Met Pro Val Leu Lys Lys Trp 255 260 265 270tgt atc ttc gag cgc gag gac ttc acc ggc gag ggg gct aag ctg cgc 1406 CysIle Phe Glu Arg Glu Asp Phe Thr Gly Glu Gly Ala Lys Leu Arg 275 280 285gac gag ctg gcc ctg gtg atc aag gac ctc gag ctg gcc tgc gac aag 1454 AspGlu Leu Ala Leu Val Ile Lys Asp Leu Glu Leu Ala Cys Asp Lys 290 295 300ttc gag gtg tcc aag caa cgc caa ctc gac cgg gaa gcc cgt acg ggc 1502 PheGlu Val Ser Lys Gln Arg Gln Leu Asp Arg Glu Ala Arg Thr Gly 305 310 315aag aag gtc agc gca cac gag ctg cat aaa acc gct ggc aaa ctg gcg 1550 LysLys Val Ser Ala His Glu Leu His Lys Thr Ala Gly Lys Leu Ala 320 325 330atg agc cgt cgt tagcccggcg acgatgcaga gcgcgcagcg cgatgagc 1600 Met SerArg Arg 335 37 338 PRT Mycobacterium tuberculosis 37 Met Ser Ala Lys LeuThr Asp Leu Gln Leu Leu His Glu Leu Glu Pro 1 5 10 15 Val Val Glu LysTyr Leu Asn Arg His Leu Ser Met His Lys Pro Trp 20 25 30 Asn Pro His AspTyr Ile Pro Trp Ser Asp Gly Lys Asn Tyr Tyr Ala 35 40 45 Leu Gly Gly GlnAsp Trp Asp Pro Asp Gln Ser Lys Leu Ser Asp Val 50 55 60 Ala Gln Val AlaMet Val Gln Asn Leu Val Thr Glu Asp Asn Leu Pro 65 70 75 80 Ser Tyr HisArg Glu Ile Ala Met Asn Met Gly Met Asp Gly Ala Trp 85 90 95 Gly Gln TrpVal Asn Arg Trp Thr Ala Glu Glu Asn Arg His Gly Ile 100 105 110 Ala LeuArg Asp Tyr Leu Val Val Thr Arg Ser Val Asp Pro Val Glu 115 120 125 LeuGlu Lys Leu Arg Leu Glu Val Val Asn Arg Gly Phe Ser Pro Gly 130 135 140Gln Asn His Gln Gly His Tyr Phe Ala Glu Ser Leu Thr Asp Ser Val 145 150155 160 Leu Tyr Val Ser Phe Gln Glu Leu Ala Thr Arg Ile Ser His Arg Asn165 170 175 Thr Gly Lys Ala Cys Asn Asp Pro Val Ala Asp Gln Leu Met AlaLys 180 185 190 Ile Ser Ala Asp Glu Asn Leu His Met Ile Phe Tyr Arg AspVal Ser 195 200 205 Glu Ala Ala Phe Asp Leu Val Pro Asn Gln Ala Met LysSer Leu His 210 215 220 Leu Ile Leu Ser His Phe Gln Met Pro Gly Phe GlnVal Pro Glu Phe 225 230 235 240 Arg Arg Lys Ala Val Val Ile Ala Val GlyGly Val Tyr Asp Pro Arg 245 250 255 Ile His Leu Asp Glu Val Val Met ProVal Leu Lys Lys Trp Cys Ile 260 265 270 Phe Glu Arg Glu Asp Phe Thr GlyGlu Gly Ala Lys Leu Arg Asp Glu 275 280 285 Leu Ala Leu Val Ile Lys AspLeu Glu Leu Ala Cys Asp Lys Phe Glu 290 295 300 Val Ser Lys Gln Arg GlnLeu Asp Arg Glu Ala Arg Thr Gly Lys Lys 305 310 315 320 Val Ser Ala HisGlu Leu His Lys Thr Ala Gly Lys Leu Ala Met Ser 325 330 335 Arg Arg 3824 DNA Artificial Sequence Description of Artificial Sequence primer 38cacggtacca agctttctag caga 24 39 53 DNA Artificial Sequence Descriptionof Artificial Sequence primer 39 gtcagtggtg gtggtggtgg tgtctagaagtactggatcc gaaaactacc tcg 53 40 27 DNA Artificial Sequence Descriptionof Artificial Sequence primer 40 tagctgcagg gatccatgac gtcggct 27 41 21DNA Artificial Sequence Description of Artificial Sequence primer 41gtgtctagac ttgtactcat g 21 42 33 DNA Artificial Sequence Description ofArtificial Sequence primer 42 gggtctagaa cgacggctca tcgccagttt gcc 33 4333 DNA Artificial Sequence Description of Artificial Sequence primer 43cccggatcca tgtcagccaa gctgaccgac ctg 33 44 76 DNA Artificial SequenceDescription of Artificial Sequence DNA construct 44 taagagaaag ggagtccacatg ccc gag gta gtt ttc gga tcc agt act tct 52 Met Pro Glu Val Val PheGly Ser Ser Thr Ser 1 5 10 aga cac cac cac cac cac cac tga 76 Arg HisHis His His His His 15 45 18 PRT Artificial Sequence Description ofArtificial Sequence amino acid sequence encoded by DNA construct 45 MetPro Glu Val Val Phe Gly Ser Ser Thr Ser Arg His His His His 1 5 10 15His His

What is claimed is:
 1. A method for screening a molecule capable ofinhibiting the growth or survival of a mycobacteria species, said methodcomprising: a) contacting the molecule with a strain of mycobacteriaspecies comprising an active DES protein, or a DES like protein, or avector comprising a first polynucleotide sequence encoding an active DESprotein, or a vector comprising a second polynucleotide sequenceencoding an active site of the DES protein; b) measuring the growth ofsaid mycobacteria strain; and c) identifying the molecule that inhibitsthe growth of said mycobacteria strain.
 2. The method of claim 1,wherein the mycobacteria species is Mycobacterium tuberculosis.
 3. Themethod of claim 2, wherein the DES protein is a recombinant DES protein.4. The method of claim 3, wherein the recombinant DES protein isobtained from a recombinant mycobacterium host cell.
 5. The method ofclaim 4, wherein the recombinant DES protein is prepared by a processcomprising: a) transforming a mycobacterium host cell with a recombinantexpression vector, wherein the recombinant expression vector comprises apolynucleotide encoding said recombinant DES protein and a regulatory orpromoter sequence that functions in mycobacteria; and b) purifying saidrecombinant DES protein from said transformed mycobacterium host cell.6. The method of claim 5, wherein the recombinant expression vector hasbeen constructed from the pJAM2 plasmid.
 7. The method of claim 6,wherein the recombinant expression vector is pJAM21.
 8. A plasmidselected from the group consisting of pJAM2 and pJAM21.
 9. A recombinantmycobacterium host cell, comprising a plasmid according to claim
 8. 10.The recombinant mycobacterium host cell according to claim 9, whereinthe plasmid is pJAM2.
 11. The recombinant mycobacterium host cellaccording to claim 9, wherein the plasmid is pJAM21.
 12. A moleculecapable of inhibiting the growth or survival of Mycobacteriumtuberculosis, wherein said molecule has been screened according to themethod of claim 2 or wherein said molecule blocks the active site of theDES protein or modulates the expression of the des gene in vivo or invitro.
 13. A molecule capable of inhibiting the growth or survival ofMycobacterium tuberculosis, wherein said molecule has been screenedaccording to the method of claim
 3. 14. A molecule capable of inhibitingthe growth or survival of Mycobacterium tuberculosis, wherein saidmolecule has been screened according to the method of claim
 4. 15. Amolecule capable of inhibiting the growth or survival of Mycobacteriumtuberculosis, wherein said molecule has been screened according to themethod of claim
 5. 16. A molecule capable of inhibiting the growth orsurvival of Mycobacterium tuberculosis, wherein said molecule has beenscreened according to the method of claim
 6. 17. A molecule capable ofinhibiting the growth or survival of Mycobacterium tuberculosis, whereinsaid molecule has been screened according to the method of claim
 7. 18.A method according to claim 17, wherein the active site of the DESprotein comprises a first amino acid sequence DEXXH (SEQ ID NO:4) or asecond amino acid sequence EEXXH (SEQ ID NO:3), wherein X can representany amino acid.
 19. A purified DES polypeptide in monomeric or dimericform, comprising an amino acid sequence DEXXHEEXXH (SEQ ID NO:5),wherein X can represent any amino acid.
 20. A purified DES polypeptidein monomeric or dimeric form, comprising an amino acid sequence DEXXH(SEQ ID NO:4), wherein X can represent any amino acid.
 21. A purifiedpolypeptide according to claim 20, further comprising a second aminoacid sequence EEXXH (SEQ ID NO:3), wherein the amino acid sequence DEXXH(SEQ ID NO:4) and the second amino acid sequence EEXXH (SEQ ID NO:3) areseparated by at least 10 to 100 amino acids.
 22. A purified polypeptideaccording to claim 21, wherein the amino acid sequence DEXXH (SEQ IDNO:4)and the second amino acid sequence EEXXH (SEQ ID NO:3) areseparated by 85 amino acids.
 23. A purified polypeptide according toclaim 22, further comprising a second amino acid sequence EEXXH (SEQ IDNO:3), wherein the polypeptide has between 10 to 100 amino acids.
 24. Acomposition for the treatment of mycobacteria infection, comprising amolecule capable of inhibiting the growth of the survival ofmycobacteria strain by inhibition of DES protein activity, wherein saidmolecule has been screened according to claim
 1. 25. A method forscreening a molecule capable of inhibiting the growth of mycobacterialspecies, said method comprising: a) contacting the molecule with apurified stearoyl or acyl ACP desaturase; and b) measuring theinhibition of the enzyme activity compared to an untreated enzyme.
 26. Amethod for screening a molecule capable of inhibiting the growth orsurvival of a mycobacteria species selected from the group consisting ofMycobacterium tuberculosis and Mycobacterium leprae, said methodcomprising: a) contacting the molecule with a purified desaturaseprotein; b) detecting the catalytic activity of the desaturase protein;and c) identifying the molecule that reduces the catalytic activity ofthe desaturase protein, as compared to the catalytic activity of thepurified desaturase protein that is not contacted with the molecule. 27.The method of claim 26, wherein the mycobacteria species isMycobacterium tuberculosis.
 28. The method of claim 27, wherein thepurified desaturase protein is a recombinant desaturase protein.
 29. Themethod of claim 28, wherein the purified recombinant desaturase proteinis obtained from a recombinant mycobacterium host cell.
 30. A moleculecapable of inhibiting or decreasing the growth or survival of amycobacteria species, wherein said molecule has been screened accordingto claim 26.